Conversion Of Violaxanthin Research Articles

Combined high light and salt stress enhances accumulation of PsbS and zeaxanthin in Chlamydomonas reinhardtii.

The performance and acclimation strategies of Chlamydomonas reinhardtii under stress conditions are typically studied in response to single stress factors. Under natural conditions, however, organisms rarely face only one stressor at a time. Here, we investigated the impact of combined salt and high light stress on the photoprotective response of C. reinhardtii. Compared to the single stress factors, the combination of both stressors decreased the photosynthetic performance, while the activation of energy dissipation remained unaffected. However, the PsbS protein was strongly accumulated and the conversion of violaxanthin to zeaxanthin was enhanced. These results support an important photoprotective function of PsbS and zeaxanthin independently of energy dissipation under combined salt and high light stress in C. reinhardtii.

Modulation of xanthophyll cycle impacts biomass productivity in the marine microalga Nannochloropsis

Life on earth depends on photosynthetic primary producers that exploit sunlight to fix CO2 into biomass. Approximately half of global primary production is associated with microalgae living in aquatic environments. Microalgae also represent a promising source of biomass to complement crop cultivation, and they could contribute to the development of a more sustainable bioeconomy. Photosynthetic organisms evolved multiple mechanisms involved in the regulation of photosynthesis to respond to highly variable environmental conditions. While essential to avoid photodamage, regulation of photosynthesis results in dissipation of absorbed light energy, generating a complex trade-off between protection from stress and light-use efficiency. This work investigates the impact of the xanthophyll cycle, the light-induced reversible conversion of violaxanthin into zeaxanthin, on the protection from excess light and on biomass productivity in the marine microalgae of the genus Nannochloropsis. Zeaxanthin is shown to have an essential role in protection from excess light, contributing to the induction of nonphotochemical quenching and scavenging of reactive oxygen species. On the contrary, the overexpression of zeaxanthin epoxidase enables a faster reconversion of zeaxanthin to violaxanthin that is shown to be advantageous for biomass productivity in dense cultures in photobioreactors. These results demonstrate that zeaxanthin accumulation is critical to respond to strong illumination, but it may lead to unnecessary energy losses in light-limiting conditions and accelerating its reconversion to violaxanthin provides an advantage for biomass productivity in microalgae.

Xanthophyll cycles in the juniper haircap moss (Polytrichum juniperinum) and Antarctic hair grass (Deschampsia antarctica) on Livingston Island (South Shetland Islands, Maritime Antarctica)

The summer climate in Maritime Antarctica is characterised by high humidity and cloudiness with slightly above zero temperatures. Under such conditions, photosynthetic activity is temperature-limited and plant communities are formed by a few species. These conditions could prevent the operation of the photoprotective xanthophyll (VAZ) cycle as low irradiance reduces the excess of energy and low temperatures limit enzyme activity. The VAZ cycle regulates the dissipation of the excess of absorbed light as heat, which is the main mechanism of photoprotection in plants. To test whether this mechanism operates dynamically in Antarctic plant communities, we characterised pigment dynamics under natural field conditions in two representative species: the moss Polytrichum juniperinum and the grass Deschampsia antarctica. Pigment analyses revealed that the total VAZ pool was in the upper range of the values reported for most plant species, suggesting that they are exposed to a high degree of environmental stress. Despite cloudiness, there was a strong conversion of violaxanthin (V) to zeaxanthin (Z) during daytime. Conversely, the dark-induced enzymatic epoxidation back to V was not limited by nocturnal temperatures. In contrast with plants from other cold ecosystems, we did not find any evidence of overnight retention of Z or sustained reductions in photochemical efficiency. These results are of interest for modelling, remote sensing and upscaling of the responses of Antarctic vegetation to environmental challenges.

Manipulation of Environmental Stress Towards Lutein Production in Chlorella fusca Cell Culture

All carotenoids originate from a single, common precursor, phytoene. The colour of carotenoids is determinedby desaturation, isomerization, cyclization, hydroxylation and epoxidation of the 40-carbon phytoene. The conjugated double-bond structure and nature of end ring groups confer on the carotenoids properties such as colour and antioxidant activity. Algae may become major sources of carotenoids but the extent of environmental stress and genetic influences on algae carotenoid biosynthesis are poorly understood. Carotenoid biosynthesis can be influenced by many aspects and is liable to geometric isomerization with the existence of oxygen, light and heat which affect the colour degradation and oxidation. Therefore, in this study carotenoid biogenesis is investigated in cell culture of Chlorella fusca as a potential model system for rapid initiation, and extraction of carotenoids by providing stringent control of genetic, developmental and environmental factors. The value of this experimental system for investigating key factors controlling the carotenoid accumulation is then tested by assessing the effects of environmental variables, such as drought stress, light intensity, nutrient strength and media formulation on carotenoid accumulation. Our findings revealed that the conversion of violaxanthin to lutein is due to irradiance stress condition, nutrient strength as well as drought stress. As a result, manipulation of environmental variables will up-regulate lutein concentration. This reaction will restrict the supply of precursors for ABA biosynthesis and the algae cell culture responds by increasing carotenogenic metabolic flux to compensate for this restriction. In conclusion, selecting the appropriate algae species for the appropriate environmental conditions is not only important for yield production, but also for nutritional value quality of carotenoid.

A Key Role of Xanthophylls That Are Not Embedded in Proteins in Regulation of the Photosynthetic Antenna Function in Plants, Revealed by Monomolecular Layer Studies.

The main physiological function of LHCII (light-harvesting pigment-protein complex of photosystem II), the largest photosynthetic antenna complex of plants, is absorption of light quanta and transfer of excitation energy toward the reaction centers, to drive photosynthesis. However, under strong illumination, the photosynthetic apparatus faces the danger of photodegradation and therefore excitations in LHCII have to be down-regulated, e.g., via thermal energy dissipation. One of the elements of the regulatory system, operating in the photosynthetic apparatus under light stress conditions, is a conversion of violaxanthin, the xanthophyll present under low light, to zeaxanthin, accumulated under strong light. In the present study, an effect of violaxanthin and zeaxanthin on the molecular organization and the photophysical properties of LHCII was studied in a monomolecular layer system with application of molecular imaging (atomic force microscopy, fluorescence lifetime imaging microscopy) and spectroscopy (UV-Vis absorption, FTIR, fluorescence spectroscopy) techniques. The results of the experiments show that violaxanthin promotes the formation of supramolecular LHCII structures preventing dissipative excitation quenching while zeaxanthin is involved in the formation of excitonic energy states able to quench chlorophyll excitations in both the higher (B states) and lower (Q states) energy levels. The results point to a strategic role of xanthophylls that are not embedded in a protein environment, in regulation of the photosynthetic light harvesting activity in plants.

Molecular studies on structural changes and oligomerisation of violaxanthin de-epoxidase associated with the pH-dependent activation.

Violaxanthin de-epoxidase (VDE) is a conditionally soluble enzyme located in the thylakoid lumen and catalyses the conversion of violaxanthin to antheraxanthin and zeaxanthin, which are located in the thylakoid membrane. These reactions occur when the plant or algae are exposed to saturating light and the zeaxanthin formed is involved in the process of non-photochemical quenching that protects the photosynthetic machinery during stress. Oversaturation by light results in a reduction of the pH inside the thylakoids, which in turn activates VDE and the de-epoxidation of violaxanthin. To elucidate the structural events responsible for the pH-dependent activation of VDE, full length and truncated forms of VDE were studied at different pH using circular dichroism (CD) spectroscopy, crosslinking and small angle X-ray scattering (SAXS). CD spectroscopy showed the formation of α-helical coiled-coil structure, localised in the C-terminal domain. Chemical crosslinking of VDE showed that oligomers were formed at low pH, and suggested that the position of the N-terminal domain is located near the opening of lipocalin-like barrel, where violaxanthin has been predicted to bind. SAXS was used to generate models of monomeric VDE at high pH and also a presumably dimeric structure of VDE at low pH. For the dimer, the best fit suggests that the interaction is dominated by one of the domains, preferably the C-terminal domain due to the lost ability to oligomerise at low pH, shown in earlier studies, and the predicted formation of coiled-coil structure.

Violaxanthin de-epoxidase disulphides and their role in activity and thermal stability.

Violaxanthin de-epoxidase (VDE) catalyses the conversion of violaxanthin to zeaxanthin at the lumen side of the thylakoids during exposure to intense light. VDE consists of a cysteine-rich N-terminal domain, a lipocalin-like domain and a negatively charged C-terminal domain. That the cysteines are important for the activity of VDE is well known, but in what way is less understood. In this study, wild-type spinach VDE was expressed in E. coli as inclusion bodies, refolded and purified to give a highly active and homogenous preparation. The metal content (Fe, Cu, Ni, Mn, Co and Zn) was lower than 1 mol% excluding a metal-binding function of the cysteines. To investigate which of the 13 cysteines that could be important for the function of VDE, we constructed mutants where the cysteines were replaced by serines, one by one. For 12 out of 13 mutants the activity dropped by more than 99.9 %. A quantification of free cysteines showed that only the most N-terminal of these cysteines was in reduced form in the native VDE. A disulphide pattern in VDE of C9–C27, C14–C21, C33–C50, C37–C46, C65–C72 and C118–C284 was obtained after digestion of VDE with thermolysin followed by mass spectroscopy analysis of reduced versus non-reduced samples. The residual activity found for the mutants showed a variation that was consistent with the results obtained from mass spectroscopy. Reduction of the disulphides resulted in loss of a rigid structure and a decrease in thermal stability of 15 °C.

The potato carotenoid cleavage dioxygenase 4 catalyzes a single cleavage of β-ionone ring-containing carotenes and non-epoxidated xanthophylls

Down-regulation of the potato carotenoid cleavage dioxygenase 4 (StCCD4) transcript level led to tubers with altered morphology and sprouting activity, which also accumulated higher levels of violaxanthin and lutein leading to elevated carotenoid amounts. This phenotype indicates a role of this enzyme in tuber development, which may be exerted by a cleavage product. In this work, we investigated the enzymatic activity of StCCD4, by expressing the corresponding cDNA in carotenoid accumulating Escherichia coli strains and by performing in vitro assays with heterologously expressed enzyme. StCCD4 catalyzed the cleavage of all-trans-β-carotene at the C9′–C10′ double bond, leading to β-ionone and all-trans-β-apo-10′-carotenal, both in vivo and in vitro. The enzyme also cleaved β,β-cryptoxanthin, zeaxanthin and lutein either at the C9′–C10′ or the C9–C10 double bond in vitro. In contrast, we did not observe any conversion of violaxanthin and only traces of activity with 9-cis-β-carotene, which led to 9-cis-β-apo-10′-carotenal. Our data indicate that all-trans-β-carotene is the likely substrate of StCCD4 in planta, and that this carotene may be precursor of an unknown compound involved in tuber development.

Positive feedback regulation of a Lycium chinense-derived VDE gene by drought-induced endogenous ABA, and over-expression of this VDE gene improve drought-induced photo-damage in Arabidopsis

Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under light stress. We have cloned a VDE gene (LcVDE) from Lycium chinense, a deciduous woody perennial halophyte, which can grow in a large variety of soil types. The amino acid sequence of LcVDE has high homology with VDEs in other plants. Under drought stress, relative expression of LcVDE and the de-epoxidation ratio (Z+0.5A)/(V+A+Z) increased rapidly, and non-photochemical quenching (NPQ) also rose. Interestingly, these elevations induced by drought stress were reduced by the topical administration of abamine SG, a potent ABA inhibitor via inhibition of NCED in the ABA synthesis pathway. Until now, little has been done to explore the relationship between endogenous ABA and the expression of VDE genes. Since V serves as a common precursor for ABA, these data support the possible involvement of endogenous ABA in the positive feedback regulation of LcVDE gene expression in L. chinense under drought stress. Moreover, the LcVDE may be involved in modulating the level of photosynthesis damage caused by drought stress. Furthermore, the ratio of (Z+0.5A)/(V+A+Z) and NPQ increased more in transgenic Arabidopsis over-expressing LcVDE gene than the wild types under drought stress. The maximum quantum yield of primary photochemistry of PSII (Fv/Fm) in transgenic Arabidopsis decreased more slowly during the stressed period than that in wild types under the same conditions. Furthermore, transgenic Arabidopsis over-expressing LcVDE showed increased tolerance to drought stress.

Photosynthesis in extreme environments: responses to different light regimes in the Antarctic alga Koliella antarctica.

Antarctic algae play a fundamental role in polar ecosystem thanks to their ability to grow in an extreme environment characterized by low temperatures and variable illumination. Here, for prolonged periods, irradiation is extremely low and algae must be able to harvest light as efficiently as possible. On the other side, at low temperatures even dim irradiances can saturate photosynthesis and drive to the formation of reactive oxygen species. Colonization of this extreme environment necessarily required the optimization of photosynthesis regulation mechanisms by algal organisms. In order to investigate these adaptations we analyzed the time course of physiological and morphological responses to different irradiances in Koliella antarctica, a green microalga isolated from Ross Sea (Antarctica). Koliella antarctica not only modulates cell morphology and composition of its photosynthetic apparatus on a long-term acclimation, but also shows the ability of a very fast response to light fluctuations. Koliella antarctica controls the activity of two xanthophyll cycles. The first, involving lutein epoxide and lutein, may be important for the growth under very low irradiances. The second, involving conversion of violaxanthin to antheraxanthin and zeaxanthin, is relevant to induce a fast and particularly strong non-photochemical quenching, when the alga is exposed to higher light intensities. Globally K. antarctica thus shows the ability to activate a palette of responses of the photosynthetic apparatus optimized for survival in its natural extreme environment.

Overexpression of tomato GDP-L-galactose phosphorylase gene in tobacco improves tolerance to chilling stress.

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco. Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-L-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H(2)O(2), demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-L-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.

Molecular Cloning and Characterization of Violaxanthin De-Epoxidase (CsVDE) in Cucumber

Violaxanthin de-epoxidase (VDE) plays an important role in protecting the photosynthetic apparatus from photo-damage by dissipating excessively absorbed light energy as heat, via the conversion of violaxanthin (V) to intermediate product antheraxanthin (A) and final product zeaxanthin (Z) under high light stress. We have cloned a violaxanthin de-epoxidase gene (CsVDE) from cucumber. The amino acid sequence of CsVDE has high homology with VDEs in other plants. RT-PCR analysis and histochemical staining show that CsVDE is expressed in all green tissues in cucumber and Arabidopsis. Using GFP fusion protein and immunogold labeling methods, we show that CsVDE is mainly localized in chloroplasts in cucumber. Under high light stress, relative expression of CsVDE and the de-epoxidation ratio (A+Z)/(V+A+Z) is increased rapidly, and abundance of the gold particles was also increased. Furthermore, CsVDE is quickly induced by cold and drought stress, reaching maximum levels at the 2nd hour and the 9th day, respectively. The ratio of (A+Z)/(V+A+Z) and non-photochemical quenching (NPQ) is reduced in transgenic Arabidopsis down-regulated by the antisense fragment of CsVDE, compared to wild type (WT) Arabidopsis under high light stress. This indicates decreased functionality of the xanthophyll cycle and increased sensitivity to photoinhibition of photosystem II (PSII) in transgenic Arabidopsis under high light stress.

Light-induced decrease of reflectance provides an insight in the photoprotective mechanisms of ripening apple fruit

The magnitude of real-time changes in reflectance of ripening apple ( Malus × domestica Borkh., cv. Antonovka) fruit induced by PAR irradiation (500 μE m −2 s −1) was monitored together with pigment composition and non-photochemical dissipation of absorbed light energy. In fruit with high chlorophyll content irradiation induced, within 180 s, a notable decrease of reflectance in the bands centred at 520, 692 and 740 nm. These changes were strongly inter-correlated and exhibited tight relationships with chlorophyll fluorescence intensity, violaxanthin de-epoxidation, NPQ, qN and qP. The decrease of the reflectance in the blue-green is suggested to be related mainly with irradiation-induced conversion of violaxanthin to zeaxanthin whereas the decrease of the reflectance in the red and NIR was obviously due to the quenching of chlorophyll fluorescence. Based on these findings, spectral index AVI ( Apple Violaxanthin cycle Index) for non-destructive assessment of violaxanthin cycle operation in apple was developed. According to the results of violaxanthin cycle monitoring with the use of AVI, its activity as well as non-photochemical quenching declined in the course of apple ripening on the background of the increase in the proportion of extrathylakoid carotenoids (mainly fatty acid-esterified xanthophylls) together with the relative amount of light intercepted by these pigments. Comparative importance of violaxanthin cycle and light screening by extrathylakoid carotenoids for photoprotection at different stages of fruit development is discussed.

Enhancement of susceptivity to photoinhibition and photooxidation in rice chlorophyll b-less mutants

Two rice chlorophyll (Chl) b-less mutants (VG28-1, VG30-5) and the respective wild type (WT) plant (cv. Zhonghua No. 11) were analyzed for the changes in Chl fluorescence parameters, xanthophyll cycle pool, and its de-epoxidation state under exposure to strong irradiance, SI (1 700 µmol m−2 s−1). We also examined alterations in the chloroplast ultrastructure of the mutants induced by methyl viologen (MV) photooxidation. During HI (0–3.5 h), the photoinactivation of photosystem 2 (PS2) appeared earlier and more severely in Chl b-less mutants than in the WT. The decreases in maximal photochemical efficiency of PS2 in the dark (Fv/Fm), quantum efficiency of PS2 electron transport (ΦPS2), photochemical quenching (qP), as well as rate of photochemistry (Prate), and the increases in de-epoxidation state (DES) and rate of thermal dissipation of excitation energy (Drate) were significantly greater in Chl b-mutants compared with the WT plant. A relatively larger xanthophyll pool and 78–83 % conversion of violaxanthin into antheraxanthin and zeaxanthin in the mutants after 3.5 h of HI was accompanied with a high ratio of inactive/total PS2 (0.55–0.73) and high 1–qP (0.57–0.68) which showed that the activities of the xanthophyll cycle were probably insufficient to protect the photosynthetic apparatus against photoinhibition. No apparent difference of chloroplast ultrastructure was found between Chl b-less mutants and WT plants grown under low, LI (180 µmol m−2 s−1) and high, HI (700 µmol m−2 s−1) irradiance. However, swollen chloroplasts and slight dilation of thylakoids occurred in both mutants and the WT grown under LI followed by MV treatment. These typical symptoms of photooxidative damage were aggravated as plants were exposed to HI. Distorted and loose scattered thylakoids were observed in particular in the Chl b-less mutants. A greater extent of photoinhibition and photooxidation in these mutants indicated that the susceptibility to HI and oxidative stresses was enhanced in the photosynthetic apparatus without Chl b most likely as a consequence of a smaller antenna size.

Increased hydrogen photoproduction by means of a sulfur-deprived Chlamydomonas reinhardtii D1 protein mutant

Abstract The photoproduction of H 2 was studied in a sulfur-deprived Chlamydomonas reinhardtii D1 mutant that carried a double amino acid substitution. The leucine residue L159 was replaced by isoleucine, and the asparagine N230 was replaced by tyrosine (L159I-N230Y). Phenotypic characterization of the mutant showed some interesting features compared to its wild type, namely: (1) a lower chlorophyll content; (2) a higher photosynthetic capacity and higher relative quantum yield of photosynthesis; (3) a higher respiration rate; (4) a very high conversion of violaxanthin to zeaxanthin during H 2 production; (5) a prolonged period of H 2 production. In standard conditions, the mutant produced more than 500 ml of H 2 , that is, more than one order of magnitude greater than its wild type, and about 5-times greater than the CC124 strain that was used for comparison. The better performance of the mutant was mainly the result of a longer production period. Biogas produced contained up to 99.5% H 2 .

Research note: Irradiance of photosynthetically active radiation determines ultraviolet‐susceptibility of photosynthesis in Ulva lactuca L.(Chlorophyta)

SUMMARYThe respective ratio of photosynthetically active to ultraviolet radiation is of crucial importance to results obtained in ultraviolet (UV)‐research on photoautotrophic organisms. Specimens of the green macroalga Ulva lactuca L. were exposed to a constant irradiance of UV‐radiation at increasing irradiances of photosynthetically active radiation (PAR). The effects of experimental irradiance and spectral composition on photoinhibition of photosynthesis and its recovery were monitored by chlorophyll fluorescence measurements and the activity of the xanthophyll cycle was assessed by high performance liquid chromatography‐(HPLC) based pigment analysis. Results indicate a UV‐induced delay in recovery from PAR‐induced photoinhibition and a deceleration of violaxanthin conversion within the xanthophyll cycle due to the presence of UV‐radiation. Also the concentration of the protective pigment lutein increased considerably and could be indicative of the existence of an additional light‐protective mechanism, as, for example, the lutein‐epoxid cycle in Ulva. In total, results clearly show that the extent of UV‐induced inhibition of photosynthesis to be found in UV‐exposure experiments is highly dependent on the irradiance of background photosynthetically active radiation: with increasing irradiance of PAR the UV‐effects were diminished. Exemplified by the green algae Ulva lactuca this study demonstrates the crucial importance of the ratios of PAR:UV applied in UV‐research, particularly when conducting laboratory experiments in an ecological context.

Laurdan fluorescence spectroscopy in the thylakoid bilayer: The effect of violaxanthin to zeaxanthin conversion on the galactolipid dominated lipid environment

Laurdan (6-lauroyl-2-dimethylaminonaphthalene) fluorescence spectroscopy has been applied to probe the physical status of the thylakoid membrane upon conversion of violaxanthin to zeaxanthin. So far, only phospholipid-dominated membranes have been studied by this method and hereby we report the first use of laurdan in mono- and digalactosyldiacylglycerol-dominated membrane systems. The generalised polarisation (GP) of laurdan was used as a measure of the structural effect of xanthophyll cycle pigments in isolated spinach ( Spinacia oleracea) thylakoids and in model membrane vesicles composed of chloroplast galactolipids. Higher GP values indicate a membrane in a more ordered structure, whereas lower GP values point to a membrane in a less ordered fluid phase. The method was used to probe the effect of violaxanthin and zeaxanthin in thylakoid membranes at different temperatures. At 4, 25 and 37 °C the GP values for dark-adapted thylakoids in the violaxanthin-form were 0.55, 0.28 and 0.26. After conversion of violaxanthin to zeaxanthin, at the same temperatures, the GP values were 0.62, 0.36 and 0.34, respectively. GP values increased gradually upon conversion of violaxanthin to zeaxanthin. Similar results were obtained in the liposomal systems in the presence of these xanthophyll cycle pigments. We conclude from these results that the conversion of violaxanthin to zeaxanthin makes the thylakoid membrane more ordered.

Membrane curvature stress controls the maximal conversion of violaxanthin to zeaxanthin in the violaxanthin cycle—influence of α-tocopherol, cetylethers, linolenic acid, and temperature

Zeaxanthin, an important component in protection against overexcitation in higher plants, is formed from violaxanthin by the enzyme violaxanthin de-epoxidase. We have investigated factors that may control the maximal degree of conversion in the violaxanthin cycle. The conversion of violaxanthin to zeaxanthin in isolated spinach thylakoids was followed at different temperatures and in the presence of lipid packing modifiers. The maximum degree of conversion was found to be 35%, 70% and 80% at 4 °C, 25 °C and 37 °C respectively. In the presence of membrane modifying agents, known to promote non-lamellar structures (H II), such as linolenic acid the conversion increased, and the maximal level of violaxanthin de-epoxidation obtained was close to 100%. In contrast, substances promoting lamellar phases (L α), such as α-tocopherol and 8-cetylether (C 16EO 8), only 55% and 35% of the violaxanthin was converted at 25 °C, respectively. The results are interpreted in light of the lipid composition of the thylakoid membrane, and we propose a model where a negative curvature elastic stress in the thylakoid lipid bilayer is required for violaxanthin de-epoxidase activity. In this model zeaxanthin with its longer hydrophobic stretch is proposed to promote lamellar arrangements of the membrane. As a result, zeaxanthin relieves the curvature elastic stress, which in turn leads to inactivation of violaxanthin de-epoxidase.

Effects of different irradiances on the photosynthetic process during ex-vitro acclimation of Anoectochilus plantlets

Six months old in vitro-grown Anoectochilus formosanus plantlets were transferred to ex-vitro acclimation under low irradiance, LI [60 µmol(photon) m-2 s-1], intermediate irradiance, II [180 µmol(photon) m-2 s-1], and high irradiance, HI [300 µmol(photon) m-2 s-1] for 30 d. Imposition of II led to a significant increase of chlorophyll (Chl) b content, rates of net photosynthesis (P N) and transpiration (E), stomatal conductance (g s), electron transfer rate (ETR), quantum yield of electron transport from water through photosystem 2 (ΦPS2), and activity of ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBPCO, EC 4.1.1.39). This indicates that Anoectochilus was better acclimated at II compared to LI treatment. On the other hand, HI acclimation led to a significant reduction of Chl a and b, P N, E, g s, photochemical quenching, dark-adapted quantum efficiency of open PS2 centres (Fv/Fm), probability of an absorbed photon reaching an open PS2 reaction centre (Fv'/Fm'), ETR, ΦPS2, and energy efficiency of CO2 fixation (ΦCO2/ΦPS2). This indicates that HI treatment considerably exceeded the photo-protective capacity and Anoectochilus suffered HI induced damage to the photosynthetic apparatus. Imposition of HI significantly increased the contents of antheraxanthin and zeaxanthin (ZEA), non-photochemical quenching, and conversion of violaxanthin to ZEA. Thus Anoectochilus modifies its system to dissipate excess excitation energy and to protect the photosynthetic machinery.

De-epoxidation of Violaxanthin in the Minor Antenna Proteins of Photosystem II, LHCB4, LHCB5, and LHCB6

The conversion of violaxanthin to zeaxanthin is essentially required for the pH-regulated dissipation of excess light energy in the antenna of photosystem II. Violaxanthin is bound to each of the antenna proteins of both photosystems. Former studies with recombinant Lhcb1 and different Lhca proteins implied that each antenna protein contributes specifically to violaxanthin conversion related to protein-specific affinities of the different violaxanthin binding sites. We investigated the violaxanthin de-epoxidation in the minor antenna proteins of photosystem II, Lhcb4-6. Recombinant proteins were reconstituted with different xanthophyll mixtures to study the conversion of violaxanthin at different xanthophyll binding sites in these proteins. The extent and kinetics of violaxanthin de-epoxidation were found to be dependent on the respective protein and, for each protein, also on the binding site of violaxanthin. In particular, violaxanthin bound to Lhcb4 was nearly inconvertible for de-epoxidation, whereas violaxanthin bound to Lhcb5 was fully convertible but with slow kinetics. Lhcb6 exhibited heterogeneous violaxanthin conversion characteristics, which could be assigned to different populations of reconstituted Lhcb6 complexes with respect to violaxanthin binding sites. The results support the proposed different binding affinities of violaxanthin to the three putative violaxanthin binding sites (V1, N1, and L2) in antenna proteins. Under consideration of former studies with Lhcb1 and Lhca proteins, the data imply that violaxanthin bound to the V1 and N1 binding site of antenna proteins is easily accessible for de-epoxidation in all antenna proteins, whereas violaxanthin bound to L2 is either only slowly or not convertible to zeaxanthin, depending on the respective protein.